a. Schematic representation of significantly and differentially expressed genes between individual stages of the transition from the ES to the 2-cell-like state (). Changes were considered significant if they exhibited at least 2-fold changes across cells between individual states and a P < 0.05 (Mann-Whitney U test).
b. Design of siRNA screen.
c. Representative inverted dynamics images for the negative (scramble siRNA cells “neg”; n=270 wells) and positive (p150 siRNA transfected cells “p150”; n=270 wells) controls of the screening are shown. Nine random images from one well were combined. Scale bar, 500 µm.
d, e. Representative images of the eGFP (d) and ZSCAN4 (e) immunostaining for selected hits from the screening are shown in inverted dynamics. Scale bar represents 500µm. Images correspond to the following siRNA in order, as indicated: 1/Snrpd3 2/Ring1b 3/Pcgf6 4/Dmap1 5/Ep400 6/Snrpb 7/Snrpd1 8/Usp7 9/Snrpe 10/Lsm6 11/Snrpd2 12/Snrp200 13/ Rif1 14/p60 15/Sf3b1 16/Prpf8 17/Psmd14 18/Aqr 19/Snrpg 20/Gmnn 21/Dnmt3b 22/Ubl5 23/Rad21 24/Recql5 25/Trrap 26/Ddx23 27/Cdcl5 28/Ncl.
f. Heatmap showing the effect on OCT4, ZSCAN4 and 2C::eGFP expression of the top 50 hits of the screening, ranked according to the percentage of 2-cell-like-cells induced upon siRNA. Data are combined from the primary and the secondary validation screening, and includes additional hits that were tested during the secondary screening. 2-cell-like-cells are defined as cells that are positive for eGFP and ZSCAN4 but negative for OCT4 immunostaining.
g. Quantification of 2-cell-like-cells (e.g. 2C::eGFP positive, ZSCAN4 positive and OCT4 negative) induced upon siRNA of the top 50 targets (grey) from the primary and secondary screening. The mean values ± s.d. from triplicate wells are shown. Each dot corresponds to measurements of independent wells. Negative (NT, non-transfected; and Neg, scramble siRNA) and positive (p150) controls are shown in red.